A novel and simple two-step procedure is proposed for detecting specific point mutations in eukaryotic, viral, or prokaryotic genomes. The aim is to develop a simple diagnostic procedure of possible clinical significance that lends itself to automatization, and does not require gel electrophoresis or blotting (RFLP), thereby permitting the screening of individuals or large populations for mutations in oncogenes and for other chromosomal mutations known to be associated with specific genetic defects. The novel method will employ two specially designed oligodeoxynucleotide probes ("IIS probes") which (1) will carry a hairpin domain with recognition for one or more class-IIS restriction enzyme(s), (2) will be complementary to a long stretch (30-50 nucleotides) of either strand of target DNA, including the site of the mutation to be detected (one probe complementary to wild-type and the other to the mutant sequence; Fig. 1), and (3) will carry a 5' or 3' terminal label. In the first step of the procedure, the labeled IIS probes will be hybridized with the denatured target DNA (e.g., from the human, neoplastic or normal tissue, Drosophila, or viral DNA), the hybrids will be trapped, and any excess or the nonhybridizing IIS probe will be washed away under stringent conditions. Because the probes will be long, they will hybridize very specifically to the proper region of both the wild-type or mutant target DNA. The second step would distinguish between the wild-type and mutant DNA by treating the hybridized IIS probes with class-IIS restriction enzymes, e.g., FokI or HphI. The probes will be designed in such a manner that only in the case of matching target DNA (wild-type versus wild- type or mutant versus mutant) will a terminally labeled mono- or dinucleotide be released. Detection of a massive release of the label associated with a mono- or dinucleotide (or even trinucleotide) should be very simple, when employing any procedure to separate monomers from polymers, e.g., precipitation, adsorption, or release from nylon filters. Since the enzyme recognition site is built into the IIS probe, the method is generally applicable for detecting any mutation and is independent of the presence or absence of natural restriction sites in the target DNA, which otherwise would have to overlap the mutations to be detected. The preliminary experiments appear very promising, but plans are to test extensively (1) many class-IIS enzymes (about 15), (2) various designs of the probes, (3) various labels, and (4) various target DNA, including specific oncogen mutants.